The essential role of chromium in nutrition was first recognized by Schwarz and Mertz in 1959 (Schwarz, K. and Mertz, W., “Chromium (III) and the glucose tolerance factor.” Archs Biochem. Biophys. 85:292 (1959)). These researchers observed that rats fed torula yeast developed glucose intolerance. However, rats fed brewer's yeast did not develop this condition. A substance present in the brewer's yeast, but not in torula yeast was termed glucose tolerance factor (GTF). Later it was demonstrated that the active ingredient in GTF is chromium (III). Since these early observations, numerous avenues of research were initiated to better understand the nutritional role of chromium. Although much is now known about the role of chromium in human and animal nutrition, there is much that is not known and many of the effects of chromium in human disease are still controversial and not well documented. Recently, several reviews have been published that summarize the current state of knowledge regarding the role of chromium in nutrition. (“Chromium as a Supplement”, Henry C. Lukaski, Ann Rev Nutr. 19:279(1999); “Chromium, Glucose Intolerance and Diabetes”, Richard A. Anderson, Journal of the American College of Nutrition, 17, 548(1998); “The Biochemistry of Chromium”, John B. Vincent, J. Nutr. 130: 715(2000); “Quest for the Molecular Mechanism of Chromium Action and its Relationship to Diabetes”, John B. Vincent, Nutrition Reviews, 58:67(2000)).
The exact nature of the Glucose Tolerance Factor originally proposed in 1959 remains elusive. A chromium-containing material that potentiated glucose metabolism was partially purified from acid-hydrolyzed Brewer's yeast and porcine kidney. The material from yeast received the most attention and was commonly referred to as yeast GTF. It was reported that chromium in yeast GTF was absorbed more readily than inorganic chromium sources. Further, it was proposed that yeast GTF is composed of chromium (III) ions, nicotinic acid, glycine, glutamic acid and cysteine. (“Preparation of chromium-containing material of glucose tolerance factor activity from Brewer's yeast extracts and by synthesis, E. W. Toepfer”, W. Mertz, M. M. Polansky et al., J Agric Food Chem, 25:162(1977)). The proposed composition of the yeast GTF remains controversial and its isolation was not reproducible in other laboratories. Additionally, it has been proposed that the isolated yeast GTF may be an artifact produced by acid hydrolysis of special chromium binding proteins. (“Is glucose tolerance factor an artifact produced by acid hydrolysis of low-molecular-weight, chromium binding substance?” K. H. Sumrall and J. B. Vincent, Polyhedron, 16: 4171(1997)).
Recently, some progress was made towards understanding the molecular basis of the action of chromium in regulating carbohydrate and lipid metabolism. A peptide known as low-molecular-weight chromium-binding substance (LMWCr) has been isolated and is believed to play a critical role in modulating the action of insulin on its receptors. This peptide appears to be widely distributed in mammalian tissues and has been isolated from a number of sources. LMWCr is composed of glycine, cysteine, glutamic acid and aspartic acid. Glutamic and aspartic acids represent more than half the amino acid residues. The peptide is 1500 Dalton and binds four chromium ions. It is present in tissues primarily in its metal-free form. The amino acid sequence of this protein and the crystal structure of its complex with chromium are not yet known. (“The Biochemistry of Chromium”, J. B. Vincent, J. Nutr. 130:715(2000)). It appears that LMWCr-bound chromium is present primarily in the form of anion bridged multinuclear chromium-carboxylate assembly. (“Synthetic Models for Low-Molecular-Weight Chromium-Binding Substance: Synthesis and characterization of Oxo-Bridged Tetranuclear Chromium (III) Assemblies”, Truitt Ellis et al, Inorg. Chem., 33: 5522(1994)). A synthetic multinuclear chromium assembly was found to activate the insulin receptor activity similar to that observed with the LMWCr. (“Synthetic Multinuclear Chromium Assembly Activates Insulin Receptor Kinase Activity: Functional Model for Low-Molecular-Weight Chromium-Binding Substance”, C. M. Davis et al, Inorg. Chem., 36:5316(1997)).
The recognition that yet an unidentified complex of chromium (III) and organic ligand(s) is responsible for modulating carbohydrate and lipid metabolism has generated significant interest in developing novel chromium containing compounds for use in human and animal nutrition. Numerous patents have been issued describing compounds that contain chromium bound to a variety of ligands. In 1975 a patent was issued to one of the inventors on this application disclosing 1:1 and 1:2 Chromium, Alpha Amino Acid Complex Salts (U.S. Pat. No. 3,925,433). These complex salts exist as ion pairs in which the cation is composed of a complex of the chromium (III) ion with one or two molecules of an alpha amino acid. The cation carries either a 1+ or a 2+depending on the number of amino acid molecules forming the complex. The counter ion (anion) may be chloride, sulfate or acid sulfate. Essential metal complexes of L-methionine, including 1:1 chromium-L-methionine complexes are disclosed in U.S. Pat. No. 5,278,329. Metal complexes of amino acids obtained by hydrolysis of proteins, including chromium-amino acid complexes are described in U.S. Pat. No. 5,698,724.
A method for obtaining concentrated glucose tolerance factor from Brewer's yeast was described in U.S. Pat. No. 4,343,905 issued in 1982. Other patents were issued since describing methods for obtaining yeast or yeast derivatives possessing biological activities in modulating carbohydrate or lipid metabolism, e.g. U.S. Pat. Nos. 4,348,483; 6,140,107; 6,159,466 and 6,248,323.
The use of the previously known compound, Chromium Acetylacetonate as a dietary supplement and pharmaceutical agent is described in U.S. Pat. No. 4,571,391. This water insoluble compound is heat stable, very stable to acids and slightly basic pH solutions. Chromium acetylacetonate is reported to be rapidly absorbed from the gastrointestinal tract after oral administration and is effective in potentiating insulin effects on glucose metabolism.
Dietary supplementation with essential metal picolinate, including chromium picolinate was first disclosed in U.S. Pat. No. 4,315,927 that was reissued on Jul. 7, 1992 as Re 33,988. In U.S. Pat. No. 4,315,927 the preparation of chromium picolinate was described (Example 4). In Re 33,988 specific claims are made to cover picolinate complexes of chromium, cobalt, copper and manganese in addition to zinc and ferrous that were covered in U.S. Pat. No. 4,315,927. A method for producing chromium picolinate complex is described in U.S. Pat. No. 5,677,461. The uses of chromium picolinate in the treatment and prevention of various diseases are disclosed in a number of patents including U.S. Pat. Nos. 5,087,623; 5,087,624; 5,175,156 and 6,329,361 B1. Compositions containing chromium picolinate and the uses of these compositions are described in U.S. Pat. Nos. 5,614,553; 5,929,066; 6,093,711; 6,136,317; 6,143,301; 6,251,888 B1 and 6,251,889 B1.
Chromium nicotinate, described as “GTF Chromium Material” and methods for its preparation were disclosed in U.S. Pat. Nos. 4,923,855 and 5,194,615. The use of chromium nicotinate for lowering blood lipid levels is described in U.S. Pat. No. 4,954,492. Compositions containing chromium nicotinate and their uses are disclosed in several patents including U.S. Pat. Nos. 5,905,075; 5,948,772; 5,980,905; 6,100,250; 6,100,251 and 6,323,192.
Pharmaceutical insulin-potentiating Cr (III) complexes possessing GTF-like activity are disclosed in U.S. Pat. No. 5,266,560. These complexes are composed of Cr (III), nicotinic acid or one of its derivatives and glutathione (a peptide containing L-glutamic acid, L-cysteine and glycine). The insulin potentiating activity of these complexes on glucose transport in isolated adipocytes in vitro is described and compared to that of similar complexes previously reported in the literature.
The use of metal proprionates, including chromium proprionate is disclosed in U.S. Pat. Nos. 5,707,679 and 6,303,158 B1. A composition containing chromium salts of short chain fatty acids and its use in animal nutrition is described in U.S. Pat. No. 5,846,581. Methods for producing metal carboxylate for use as animal feed supplements are described in U.S. Pat. Nos. 5,591,878 and 5,795,615.
Bioavailable chelates of creatine and essential metals, including chromium are described in U.S. Pat. No. 6,114,379. This patent claims a creatine-chromium complexes containing from 1-3 equivalents of the ligand for each chromium ion.
The use as a nutritional supplement or in the treatment of medical conditions of a previously known tri-nuclear chromium (III) complex is described in U.S. Pat. No. 6,149,948 and 6,197,816 B1. The complex is represented by the formula [Cr3O(O2CCH2CH3)6(H2O)3]+. The biological effects of the complex on a number of enzymes involved in carbohydrate and lipid metabolism are described in these patents. A method for the isolation of bovine low-molecular weight Cr-binding substance and its use are described in U.S. Pat. No. 5,872,102. This substance enhanced the insulin-activated uptake of glucose by rat adipocyts and activated rat adipocytic membrane tyrosine kinase and phosphotyrosine phosphatase activities.
Several shortcomings have been identified that limit the effectiveness of the various chromium complexes described in the literature. Chromium picolinate is the most popular of the commercially available chromium complexes. However this compound has limited water solubility and some recent studies questioned its safety. Although the lack of toxicity of chromium chloride and chromium picolinate has been demonstrated in rats (“Lack of Toxicity of Chromium Chloride and Chromium Picolinate in Rats”, Anderson et al, J. Amer. Coll. Nutr. 16: 273(1997), recent studies reported that chromium picolinate cleaves DNA and produces chromosome damage in Chinese hamster ovary cells. (“The Nutritional Supplement Chromium (III) Tris (picolinate) Cleaves DNA”, J. K. Speetjens et al, Chem. Res. Toxicol. 12:483(1999) and “Chromium (III) picolinate produces chromosome damage in Chinese hamster ovary cells”, D. M. Steams, FASEB J., 9:1643(1995)). A study of the in vivo distribution of chromium(III) picolinate in rats concluded that the short lifetime of this compound in vivo minimizes the potential toxic effects of this dietary supplement. (“In Vivo Distribution of Chromium from Chromium Picolinate in Rats and Implications for the Safety of the Dietary Supplement”, D. D. D. Hepburn and J. B. Vincent, Chem. Res. Toxicol., 15:93(2002)). For these reasons, it is clear that an alternative source of dietary chromium that is soluble, bioavailable, efficacious and safe is needed.
It is a primary objective of this invention to fulfill the above described need.
It is another objective of this invention is to provide novel 1:3 complexes of chromium (III) and alpha amino acids for use as nutritional supplement for humans and domesticated animals.
A still further objective of the invention is to provide methods for preparation of these novel complexes.
Yet another objective is to provide and describe the desirable effects of these complexes on animal performance.
An another objective of the invention is to demonstrate the lack of toxicity of the novel complexes in laboratory animals.
The structures here are 1:3 complexes of chromium (III) and alpha amino acids. The structure and properties of most of the available nutritionally relevant chromium complexes have been previously studied. For example, the mononuclear and binuclear complexes of chromium (III) picolinate have been synthesized and their structures were determined by x-ray crystallography. The reaction of chromium (III) chloride with picolinic acid in water at a pH<4.0 produced the mononuclear complex in which the ratio of metal to amino acid is 1:3 (chromium tri-picolinate). However, if the pH of the solution was >4.0, the binuclear complex was formed. The ratio of chromium to amino acid in the binuclear complex is 1:2. (“Mononuclear and Binuclear Chromium (III) Picolinate Complexes”, D. M. Steams and W. H. Armstrong, Inorg. Chem., 31:5178(1992)).
The composition and biological activity of chromium complexes of picolinic acid and nicotinic acid have also been studied. The chromium complexes formed with these pyridine carboxylic acids are different because of the differences in the structure of the two compounds. Nicotinic acid is not an alpha amino acid and hence serves as a mono- dentate ligand. It binds with chromium through the carboxylate anion and forms in and tri-nuclear complexes. Two complexes were formed between chromium and nicotinic acid, the 1:1 and 1:2. Neither complex had biological activity in the battery of tests used in this study except that the chromium dinicotinate potentiated insulin activity in rat isolated adipose tissue. Picolinic acid on the other hand is an alpha amino acid and serves as a di-dentate ligand. It binds with the chromium ion through the pyridine nitrogen and carboxyloxygen to form a stable five-member ring. Three different complexes were obtained when a solution of chromium chloride was treated with picolinic acid depending on the ratio of picolinic acid to chromium in the reaction mixture. The addition of one or two molar equivalents of picolinic acid to the chromium chloride solution caused a change in the color of the solution. Adjusting the solution to pH 7.4 with sodium hydroxide resulted in the precipitation of the complexes. These complexes were found to be homogenous by High Performance Liquid Chromatography (HPLC). When one molar equivalent of picolinic acid was used the product had the structure Cr Pic (H2O)2(OH)2. The precipitate obtained when two molar equivalents were used had the structure Cr(Pic)2(H2O)(OH).(H2O). Since these complexes were formed at pH>4 they are most likely the binuclear complexes. Neither of the two complexes had biological activity. The addition of three molar equivalents of picolinic acid to a solution of chromium chloride in water results in the formation of a red solid that precipitated from solution. This precipitate was found to be homogenous by HPLC. Analysis of the precipitate indicated that it is the chromium tripicolinate monohydrate, Cr (Pic)3.H2O. This material is most likely the mononuclear complex. This complex increased glucose uptake by rat skeletal muscle cultures in vitro. Addition of the complex to rat diet produced significant decrease in plasma glucose and prevented glycation of hemoglobin. Dietary supplementation of the chromium tripicolinate in Humans resulted in a significant increase in lean body mass in both males and females. (“Composition and Biological Activity of Chromium-Pyridine Carboxylate complexes”, G W Evans and D J Pouchnik, J. Inorg. Biochem., 49:177(1993)).
It can therefore be seen that all of the structures currently available differ from the chromium compounds of the present invention which are a different empirical formula and a different stereochemistry.